Excision of introns from precursor messenger RNA by the spliceosome is a critical step in almost all human gene expression. This process is highly regulated, integrally linked with the transcription of genes and other processing events, such as polyadenylation and nucleotide modification. A better understanding of pre-mRNA splicing will be essential to further understand mechanisms that regulate splicing, that control patterns of alternative splicing, and that contribute to development, oncogenesis and retroviral infections. The mechanism by which the spliceosome recognizes the exact sites for the chemical events and how the reactions are catalyzed are not well understood. The long-term goals of this project are to understand interactions and rearrangements between spliceosome components and the RNA ligands that are substrates for the catalytic reactions. Ample evidence argues for multiple rearrangements of factors and multiple recognition events at the branch site. Investigation of these events - which are not understood mechanistically - will elucidate interactions and rearrangements among core components and may serve as a paradigm for rearrangements in the spliceosome and in other RNP machines. This proposal focuses on mechanisms by which altered spliceosomal dynamics impact splicing fidelity. Experiments will investigate the contribution of the ATPase Prp5 to the fidelity of spliceosome assembly. As the first ATP-dependent event, this step provides a uniquely simplified system for studying the action of a spliceosomal ATPase. Further experiments will focus on the mechanism of identification of the branch site nucleophile, which will use an 'orthogonal spliceosome'system in yeast. Finally, using a genetic screen, we are investigating components of the spliceosome that strongly alter the transition from the 1st to 2nd step of splicing. This has led to a new view of the mechanism by which non-consensus splice sites are used, through alteration of the equilibrium between 1st and 2nd step conformations. These studies will help elucidate important features of spliceosome function - as these new mutants in Cef1, Prp8, and U6 snRNA have the feature of strongly altering the function of the spliceosome on mutated splice/branch sites.